Eiagshc

Total liver glutathione (GSH) and oxidized glutathione (GSSG) concentrations were determined using a Glutathione Colorimetric Detection Kit (EIAGSHC, Invitrogen). Twenty mg of frozen liver tissue was homogenized with a rod homogenizer (Polytron® PT 2500 E) in ice-cold 1 x phosphate-buffered saline (PBS) and immediately centrifuged (14,000 rpm, 10 min, 4 °C). After protein quantification using the Coomassie Plus (Bradford) Assay Kit (Thermo Scientific), the supernatant was deproteinized with 5% 5-sulfo-salicylic acid dihydrate solution (SSA). For GSSG determination, free GSH and other thiols in the samples were blocked with 2-vinylpyridine (2VP). After adding colorimetric detection reagent and reaction mixture provided with the kit, colorimetric reaction was detected at a wavelength of 405 nm using a Tecan Infinite M 200 pro plate reader. Free glutathione concentration was calculated by subtracting GSSG from GSH. Glutathione levels were normalized for protein content and expressed as μmol/g protein.

Quantification of the reduced (GSH), free GSH, and oxidized GSH (GSSG) in the hypothalamus and liver were measured using a commercial kit according to the manufacturer’s instructions (Invitrogen, Waltham, MA, USA; Cat# EIAGSHC).

Intracellular GSH/GSSG levels were measured by employing a glutathione colorimetric assay kit according to the manufacturer’s instruction (Invitrogen, Milan, Italy, catalogue number EIAGSHC).

Serum glutathione (GSH) concentration was measured using a glutathione colorimetric detection kit (Invitrogen EIAGSHC). Briefly, serum was treated with 5% SSA and incubated for 10 minutes at 4°C followed by centrifugation at 14,000 rpm for 10 minutes at 4°C. The supernatant was collected and added in a 96-well plate followed by dilution with an assay buffer. Absorbance was read at 405 nm in a microplate reader.

To measure the glutathione levels, a colorimetric detection kit (catalog num. EIAGSHC) was used (Invitrogen, Thermo Fisher Scientific, Merelbeke, Belgium). PC-3 and DU-145 were seeded 24 h prior treatment at a density of 5 × 10⁵ cells in a 100 mm² petri dish with the appropriate medium. Cells were treated either with DMSO 1 µM (negative control), MM10 1 µM or L-BSO 25 µM (positive control) for 24 h. The levels of total glutathione (GSH_tot) and oxidized glutathione (GSSG) were assessed following the manufacturer instructions. The levels of reduced glutathione (GSH) were deduced from the data collected for GSH_tot and GSSG. For each condition, protein quantification was performed using the BCA Protein Assay Kit (Pierce^TM, Thermo Fisher). The spectrophotometer used for detection was a SpectraMax M2e plate reader (Molecular Devices).

Glutathione (GSH) and oxidized glutathione (GSSG) were measured using a glutathione colorimetric detection kit (Invitrogen™, EIAGSHC) according to the manufacturer’s instructions. Briefly, NRCMs at a density of 1 × 10⁵ per cm² were seeded in a gelatin-coated 6-well culture plate. The cells were treated with vehicle (DMSO), erastin, or erastin with HC for 6 h, and then washed once with PBS. Next, the cells were harvested using a cell lifter and the cells were homogenized in ice-cold 5% 5-sulfosalicylic acid dihydrate (SSA, Sigma-Aldrich, S2130), centrifuged at 14,000 rpm for 10 min at 4 °C, and the supernatant was collected for analysis.
Heart tissues were homogenized in ice-cold phosphate buffer and then centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was collected for analysis. Total protein was quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23225).
The samples were treated with 2 VP (2-vinylpyridine) (Sigma-Aldrich, 132292) to measure GSSG. Analysis was performed according to the assay kit’s instructions, and absorbance was read at a wavelength of 405 nm (A405). Glutathione concentrations were calculated from the standard curve.